Method of treating or preventing tissue deterioration, injury or damage due to congestive heart failure

ABSTRACT

A method of treatment for treating, preventing, inhibiting or reducing tissue deterioration, injury or damage due to congestive heart failure disease, or for restoring tissue adversely affected by said disease, in a subject, includes administering to a subject an effective amount of a composition including a peptide agent including amino acid sequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2], a conservative variant thereof, or a peptide agent that stimulates production of an LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2] peptide, or a conservative variant thereof, in the tissue.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. §371 National Phase Entry Application ofPCT/US2006/028996, filed 26 Jul. 2006, and claims the benefit of U.S.Provisional application Ser. No. 60/702,269, filed 26 July 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of treating or preventingtissue deterioration, injury or damage due to congestive heart failure.

2. Description of the Background Art

Congestive heart failure (CHF) disease is manifested in an imbalance inheart pump function in which the heart fails to maintain the circulationof blood adequately. The most severe manifestation of CHF, pulmonaryedema, develops when this imbalance causes an increase in lung fluidsecondary to leakage from pulmonary capillaries into the interstitiumand alveoli of the lung.

CHF can be categorized as forward or backward ventricular failure.Backward failures is secondary to elevated systemic venous pressure,while left ventricular failure is secondary to reduced forward flow intothe aorta and systemic circulation. Furthermore, heart failure can besubdivided into systolic and diastolic dysfunction. Systolic dysfunctionis characterized by a dilated left ventricle with impairedcontractility, while diastolic dysfunction occurs in a normal or intactleft ventricle with impaired ability to relax and receive as well aseject blood.

The New York Heart Association's functional classification of CHF is oneof the most useful. Class I describes a patient who is not limited withnormal physical activity by symptoms. Class II occurs when ordinaryphysical activity results in fatigue, dyspnea, or other symptoms. ClassIII is characterized by a marked limitation in normal physical activity.Class IV is defined by symptoms at rest or with any physical activity.

CHF is summarized best as an imbalance in Starling forces or animbalance in the degree of end-diastolic fiber stretch proportional tothe systolic mechanical work expended in an ensuing contraction. Thisimbalance may be characterized as a malfunction between the mechanismsthat keep the interstitium and alveoli dry and the opposing forces thatare responsible for fluid transfer to the interstitium.

Maintenance of plasma oncotic pressure (generally about 25 mm Hg) higherthan pulmonary capillary pressure (about 7-12 mm Hg), maintenance ofconnective tissue and cellular barriers relatively impermeable to plasmaproteins, and maintenance of an extensive lymphatic system are themechanisms that keep the interstitium and alveoli dry.

Opposing forces responsible for fluid transfer to the interstitiuminclude pulmonary capillary pressure and plasma oncotic pressure. Undernormal circumstances, when fluid is transferred into the lunginterstitium with increased lymphatic flow, no increase in interstitialvolume occurs. When the capacity of lymphatic drainage is exceeded,however, liquid accumulates in the interstitial spaces surrounding thebronchioles and lung vasculature, thus creating CHF. When increasedfluid and pressure cause tracking into the interstitial space around thealveoli and disruption of alveolar membrane junctions, fluid floods thealveoli and leads to pulmonary edema.

In the U.S., more than 3 million people have CHF, and more than 400,000new patients present yearly. Prevalence of CHF is 1-2% of the generalpopulation.

Approximately 30-40% of patients with CHF are hospitalized every year.CHF is the leading diagnosis-related group among hospitalized patientsolder than 65 years. The 5-year mortality rate after diagnosis wasreported in 1971 as 60% in men and 45% in women. In 1991, data showedthe 5-year mortality rate of CHF essentially remaining unchanged, with amedian survival of 3.2 years for males and 5.4 years for females. Thismay be secondary to an aging U.S. population with declining mortalitydue to other diseases.

The most common cause of CHF death is progressive heart failure, butsudden death may account for up to 45% of all deaths. After auditingdata on 4606 patients hospitalized with CHF between 1992-1993, the totalin-hospital mortality rate was 19%, with 30% of deaths occurring fromnoncardiac causes.

There remains a need in the art for methods of treatment for treating,preventing, inhibiting or reducing tissue deterioration, injury ordamage due to congestive heart failure.

SUMMARY OF THE INVENTION

In accordance with one aspect, a method of treatment for treating,preventing, inhibiting or reducing tissue deterioration, injury ordamage due to congestive heart failure disease, or for restoring tissueadversely affected by said disease, in a subject, comprisesadministering to a subject in need of such treatment an effective amountof a composition comprising a peptide agent comprising amino acidsequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2], a conservativevariant thereof, or a stimulating agent that stimulates production of anLKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2] peptide, or aconservative variant thereof, in said tissue, so as to inhibit saidtissue deterioration, injury or damage due to congestive heart failuredisease, or restore tissue adversely affected by said disease.

DETAILED DESCRIPTION OF THE INVENTION

Congestive heart failure, also called heart failure, is a disorder inwhich the heart loses its ability to pump blood efficiently and can nolonger keep up with the normal demands placed on it. As a consequence ofconditions that damage or weaken heart muscle and cause the ventriclesto stretch (dilate), the failing pump does not move blood efficientlythroughout the circulatory system. Blood starts to back up, increasingthe pressure in the blood vessels and forcing fluid from the vesselsinto body tissues. This condition may affect the right side, the leftside or both sides of the heart. Typically, heart failure begins to fail(left-sided heart failure/left ventricular heart failure), the backpressure of blood forces fluid to collect in the lungs (pulmonaryedema). This extra fluid in the lungs makes it more difficult for theairways to expand during inhalation. Breathing becomes more difficultand shortness of breath (dyspnea) occurs. When the right side of theheart starts to fail (right-sided heart failure), there's a backpressure of blood trying to enter the heart causing fluid to collect inthe feet, ankles and lower legs. As the heart failure worsens, the upperlegs swell and eventually the abdomen collects fluid (ascites). Weightgain accompanies the fluid retention. Puffy swelling (peripheral edema)is a sigh of right heart failure, especially if the edema is pittingedema. This buildup of fluid in the lungs, legs, feet, ankles, liver andabdomen is the congestive part of heart failure.

The development of CHF is progressive and is generally a chronic,long-term condition, although it may develop suddenly following a heartattach or an inflammatory disease of the heart muscle, such as dilatedor restrictive cardiomyopathy, or the like.

Dilated (congestive) cardiomyopathy is the most common form and occursdue to enlarging and stretching (cardiac dilation) of the heart cavity,weakening the heart so it pumps abnormally. Abnormal heart rhythms(arrhythmias) and disturbances of the heart's electrical conduction mayalso occur. The cause of the condition is unknown in many cases, but itcan be caused by a virus, autoimmune diseases such as rheumatoidarthritis, and excess alcohol consumption. Additionally, blood flowsmore slowly and turbulently through an enlarged and arrhythmic heart,leading to the formation of blood clots.

Restrictive Cardiomyopathy is the least common type of cardiomyopathyand develops when damaged heart muscle is replaced by fibrous scar andfatty tissue. The right side of the heart, including the atrium mayfirst thicken and later dilate, i.e., become thinner. It may lead todisordered electrical activity and in some cases with the heart'spumping action. For the most part the ventricles become so stiff thatit's hard for them to fill with blood between contractions.

CHF becomes more common with advancing age. Risk factors includeobesity, diabetes, tobacco usage, alcohol abuse, cocaine usage.

Etiology

The most common cause of congestive heart failure is coronary arterydisease (atherosclerosis), which weakens the heart by leaving some areasof myocardium chronically deprived of oxygen rich blood and nutrients.Consequently, these areas of the heart pump less vigorously. In manycases, blood flow to myocardium is just enough to keep the muscle alivebut not functioning well. In other cases, a heart attack occurs whenblood flow is completely blocked to an area of the heart muscle. Regionsof myocardial death weaken the overall ability of the heart to pump.

Another important cause of CHF is hypertension, resulting in leftventricular heart failure. If blood pressure is high, the heart has towork harder than it should to circulate blood throughout the body. Overtime, the heart muscle may become thicker to compensate for the extrawork it must perform, but to no or little avail. In some cases the heartwill enlarge. Eventually, heart muscle may become either too stiff ortoo weak to pump blood effectively.

Other structural or functional causes of heart failure include valvularheart disease (incompetent or stenotic), congenital heart disease,dystrophinopathies, idiopathic dilated cardiomyopathy (heart muscledisease/damage/weakness of unknown origin), prolonged seriousarrhythmias, and lung disease (e.g., pulmonary arterial hypertension).

CHF Classification

Systolic heart failure: The pumping action of the left ventricle isreduced or weakened causing it to lose its ability to contractvigorously. A common clinical measurement of left ventricular functionis the ejection fraction. Patients with systolic heart failure have adecreased ejection fraction of less than 50% (usually around 40% orless). A normal ejection fraction is greater than 50%.

Diastolic heart failure: The left ventricle appears to contract normallybut it has lost its ability to relax or fill fully (i.e., it is stiff,or less compliant, when it is relaxing and filling with blood). Thisimpedes blood filing into the heart and produces backup into the lungs.Diastolic heart failure is more common in individuals over the age of75. In diastolic heart failure ejection fraction is normal.

Without being found to any specific theory, actin-sequestering peptidessuch as thymosin β4 (Tβ4 or TB4) and other agents includingactin-sequestering peptides or peptide fragments containing amino acidsequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2], or conservativevariants thereof, promote reversal or prevention of tissuedeterioration, injury or damage due to congestive heart failure disease.

Thymosin beta 4 was initially identified as a protein that isup-regulated during endothelial cell migration and differentiation invitro. Thymosin beta 4 was originally isolated from the thymus and is a43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety oftissues. Several roles have been ascribed to this protein including arole in a endothelial cell differentiation and migration, T celldifferentiation, actin sequestration, vascularization and wound healing.

In accordance with one embodiment, the invention is a method oftreatment for treating, preventing, inhibiting or reducing tissuedeterioration, injury or damage due to congestive heart failure disease,or for restoring tissue adversely affected by said disease, in asubject, comprising administering to a subject in need of such treatmentan effective amount of a composition comprising a peptide agent, whichmay be a polypeptide comprising amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO: 2], or a conservative variant thereof havingcongestive heart failure disease-inhibiting activity, preferablyThymosin β4, and/or Tβ4 isoforms, analogues or derivatives, includingKLKKTET [SEQ ID NO: 3], LKKTETQ [SEQ ID NO: 4], oxidized Tβ4, N-terminalvariants of Tβ4, C-terminal variants of Tβ4 and the like.

In preferred embodiments, the tissue deterioration, injury or damagecomprises at least one of an increase in heart size or mass, increase insize or mass of heart tissue, thickening of heart muscle, or pulmonaryedema.

Compositions which may be used in accordance with the present inventioninclude peptide agents such as Thymosin β4 (Tβ4), and/or Tβ4 isoforms,analogues or derivatives, including oxidized Tβ4, N-terminal variants ofTβ4, C-terminal variants of Tβ4 and antagonists of Tβ4, polypeptides orpeptide fragments comprising or consisting essentially of the amino acidsequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2], or conservativevariants thereof, having congestive heart failure disease-inhibitingactivity. International Application Serial No. PCT/US99/17282,incorporated herein by reference, discloses isoforms of Tβ4 which may beuseful in accordance with the present invention as well as amino acidsequence LKKTET [SEQ ID NO: 1] and conservative variants thereof, whichmay be utilized with the present invention. International ApplicationSerial No. PCT/GB99/00833 (WO 99/49883), incorporated herein byreference, discloses oxidized Thymosin β4 which may be utilized inaccordance with the present invention. Although the present invention isdescribed primarily hereinafter with respect to Tβ4 and Tβ4 isoforms, itis to be understood that the following description is intended to beequally applicable to amino acid sequence LKKTET [SEQ ID NO: 1] orLKKTNT [SEQ ID NO: 2], peptides and fragments comprising or consistingessentially of LKKTET [SEQ ID NO: 1]or LKKTNT [SEQ ID NO: 2],conservative variants thereof having congestive heart failuredisease-inhibiting activity, and/or Tβ4 isoforms, analogues orderivatives, including oxidized Tβ4, N-terminal variants of Tβ4,C-terminal variants of Tβ4 and the like.

In one embodiment, the invention provides a method of treatment fortreating, preventing, inhibiting or reducing tissue deterioration,injury or damage due to congestive heart failure disease, or forrestoring tissue adversely affected by said disease, in a subject, bycontacting heart tissue with an effective amount of a composition whichcontains a peptide agent as described herein. The contacting may bedirectly or systemically. Examples of direct administration include, forexample, contacting the tissue, by direct application or inhalation,with a solution, lotion, salve, gel, cream, paste, spray, suspension,dispersion, hydrogel, ointment, foam or oil comprising a peptide agentas described herein. Systemic administration includes, for example,intravenous, intraperitoneal, intramuscular injections of a compositioncontaining a peptide agent as described herein, in a pharmaceuticallyacceptable carrier such as water for injection.

Peptide agents for use in the invention, as described herein, may beadministered in any effective amount. For example, a peptide agent asdescribed herein may be administered in dosages within the range ofabout 0.0001-1,000,000 micrograms, more preferably in amounts within therange of about 0.1-5,000 micrograms, most preferably within the range ofabout 1-30 micrograms.

A composition in accordance with the present invention can beadministered daily, every other day, every other week, every othermonth, etc., with a single application or multiple applications per dayof administration, such as applications 2, 3, 4 or more times per day ofadministration.

Many Tβ4 isoforms have been identified and have about 70%, or about 75%,or about 80% or more homology to the known amino acid sequence of Tβ4.Such isoforms include, for example, Tβ4^(ala,) Tβ9, Tβ10, Tβ11, Tβ12,Tβ13, Tβ14 and Tβ15. Similar to Tβ4, the Tβ10 and Tβ15 isoforms havebeen shown to sequester actin. Tβ4, Tβ10 and Tβ15, as well as theseother isoforms share an amino acid sequence, LKKTET [SEQ ID NO: 1] orLKKTNT [SEQ ID NO: 2], that appears to be involved in mediating actinsequestration or binding. Although not wishing to be bound by anyparticular theory, the activity of peptide agents as described hereinmay be due, at least in part, to the anti-inflammatory and/or actinmodulating activity of such agents. Tβ4 modulates actin polymerization(e.g. β-thymosins appear to depolymerize F-actin by sequestering freeG-actin). T Tβ4's ability to modulate actin polymerization may be due toits ability to bind to or sequester actin via the LKKTET [SEQ ID NO: 1]or LKKTNT [SEQ ID NO: 2] sequence. Thus, as with Tβ4, other proteinswhich are anti-inflammatory and/or bind or sequester actin, or modulateactin polymerization, including Tβ4 isoforms having the amino acidsequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2], are likely tobe effective, alone or in a combination with Tβ4, as set forth herein.

Peptide agents as described herein, such as Tβ4 and Tβ4 isoforms,decrease inflammatory chemokine, cytokine and capase activity.

Thus, it is specifically contemplated that known Tβ4 isoforms, such asTβ4^(ala,) Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15, as well as Tβ4isoforms not yet identified, will be useful in the methods of theinvention. As such Tβ4 isoforms are useful in the methods of theinvention, including the methods practiced in a subject. The inventiontherefore further provides pharmaceutical compositions comprising Tβ4,as well as Tβ4 isoforms Tβ4^(ala,) Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 andTβ15, and a pharmaceutically acceptable carrier.

In addition, other agents or proteins having anti inflammatory activityand/or actin sequestering or binding capability, or that can mobilizeactin or modulate actin polymerization, as demonstrated in anappropriate sequestering, binding, mobilization or polymerization assay,or identified by the presence of an amino acid sequence that mediatesactin binding, such as LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2],for example, can similarly be employed in the methods of the invention.Such proteins may include gelsolin, vitamin D binding protein (DBP),profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, cappingprotein, .beta.-actinin and acumentin, for example. As such methodsinclude those practiced in a subject, the invention further providespharmaceutical compositions comprising gelsolin, vitamin D bindingprotein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin,severin, capping protein, p-actinin and acumentin as set forth herein.Thus, the invention includes the use of an polypeptide comprising theamino acid sequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2] andconservative variants thereof.

As used herein, the term “conservative variant” or grammaticalvariations thereof denotes the replacement of an amino acid residue byanother, biologically similar residue. Examples of conservativevariations include the replacement of a hydrophobic residue such asisoleucine, valine, leucine or methionine for another, the replacementof a polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like.

Tβ4 has been localized to a number of tissue and cell types and thus,agents which stimulate the production of an LKKTET [SEQ ID NO: 1] orLKKTNT [SEQ ID NO: 1] peptide such as Tβ4 or another peptide agent asdescribed herein, can be added to or comprise a composition to effectproduction a peptide agent from a tissue and/or a cell. Such stimulatingagents may include members of the family of growth factors, such asinsulin-like growth factor (IGF-1), platelet derived growth factor(PDGF), epidermal growth factor (EGF), transforming growth factor beta(TGF-β), basic fibroblast growth factor (bFGF), thymosin α1 (Tα1) andvascular endothelial growth factor (VEGF). More preferably, thestimulating agent is transforming growth factor beta (TGF-β) or othermembers of the TGF-β superfamily.

In accordance with one embodiment, subjects are treated with astimulating agent that stimulates production in the subject of a peptideagent as defined herein.

Additionally, other agents that assist in reduction of tissuedeterioration, injury or damage due to congestive heart failure disease,or restoring tissue adversely affected by said disease may be added to acomposition along with a peptide agent as described herein. For example,and not by way of limitation, a peptide agent as described herein aloneor in combination can be added in combination with any one or more ofthe following agents: antibiotics, VEGF, KGF, FGF, PDGF, TGFβ, IGF-1,IGF-2, IL-1, prothymosin α and/or thymosin α1 in an effective amount.

The invention also includes a pharmaceutical composition comprising atherapeutically effective amount of a peptide agent as described hereinin a pharmaceutically acceptable carrier. Such carriers include thoselisted herein.

The actual dosage or reagent, formulation or composition that providestreatment may depend on many factors, including the size and health of asubject. However, persons of ordinary skill in the art can use teachingsdescribing the methods and techniques for determining clinical dosagesas disclosed in PCT/US99/17282, supra, and the references cited therein,to determine the appropriate dosage to use.

Suitable formulations may include a peptide agent as described herein ata concentration within the range of about 0.001-50% by weight, morepreferably within the range of about 0.01-0.1% by weight, mostpreferably about 0.05% by weight.

The therapeutic approaches described herein involve various routes ofadministration or delivery of a peptide agent as described herein,including any conventional administration techniques (for example, butnot limited to, direct administration, local injection, inhalation, orsystemic administration), to a subject. The methods and compositionsusing or containing a peptide agent as described herein may beformulated into pharmaceutical compositions by admixture withpharmaceutically acceptable non-toxic excipients or carriers.

The invention includes use of antibodies which interact with, enhance orinhibit a peptide agent as described herein. Antibodies which consistessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known to those skilled in theart as disclosed in PCT/US99/17282, supra. The term antibody as used inthis invention is meant to include monoclonal and polyclonal antibodies.

In yet another embodiment, the invention provides a method of treating asubject by administering an effective amount of stimulating agent whichmodulates gene expression. The term “modulate” refers to inhibition orsuppression of expression when a peptide agent as described herein isover expressed, and induction of expression when a peptide agent asdescribed herein is underexpressed. The term “effective amount” meansthat amount of stimulating agent which is effective in modulating geneexpression of a peptide agent as described herein, resulting in reducingthe symptoms of tissue deterioration, injury or damage due to congestiveheart failure disease, or restoring tissue adversely affected by saiddisease. A stimulating agent which modulates gene expression of apeptide agent as described herein may be a polynucleotide, for example.The polynucleotide may be an antisense, a triplex agent, or a ribozyme.For example, an antisense directed to the structural gene region or tothe promoter region of a peptide agent as described herein may beutilized. The stimulating agent which modulates gene expression of apeptide agent as described herein may also be a small interfering RNAs(siRNAs).

In another embodiment, the invention provides a method for utilizingcompounds that modulate activity of a peptide agent as described herein.Compounds that affect activity of a peptide agent as described herein(e.g., antagonists and agonists) include peptides, peptidomimetics,polypeptides, chemical compounds, minerals such as zincs, and biologicalagents.

A method for screening for a stimulating agent as defined herein,comprises contacting heart tissue exhibiting congestive heart failuredisease, with a candidate compound; and measuring activity in saidtissue of an LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2] peptide,wherein an increase of activity of said peptide in said tissue, comparedto a level of activity of said peptide in a corresponding tissue lackingsaid candidate compound, indicates that said compound is capable ofinducing said stimulating agent.

EXAMPLE 1 Study of Thymosin β4 in Mouse Myocardial Infarction (MI) HeartFailure Model

Objective:

The objective of the present study was to evaluate the effect ofthymosin β4 in mouse MI-induced heart failure model.

Models

The procedures used in this study are exactly same as that reported inNature (432:466-472, 2004). Briefly, C57 black mice, 14 weeks old, wereused in this study. Mice were given thymosin β4 (Bachem, Lot #FTHYB40501B) (150 μg/mouse) immediately after myocardial infarction andthen every three days for 4 weeks by i.p. injection. The parameters ofthis study included cardiac morphology and function by echocardiography(echo), invasive cardiac function measurement using Millar catheter,survival rate, and histochemical analysis.

A heart attack (myocardial infarction) may rapidly lead to cardiacarrest (no heartbeat) or permanent damage of the left ventricle. If thedamage is bad enough, regions of the heart may not function properly,which may lead to heart failure. In Nature 2004 (cited above) it wasshown that TB4 was able to improve fractional shortening and ejectionfraction when administered immediately following a heart attack in themouse model. In this case the drug was administered accurately andfunctional improvements were demonstrated. Although functionalimprovements are critical for patient survival, no data were presentedto suggest that TB4 would be able to attenuate or improve outcomefollowing the significant damage that resulted from the heart attackinduced in the animal model. Consequently, it was not obvious that TB4would be able to treat a resulting heart failure that would follow inthe surviving animals.

The present study suggests that TB4 is effective in treating mammalswith heart failure/congestive heart failure (CHF). In this case TB4 wasalso administered acutely. However, in practice the drug may beadministered acutely and/or chronically following an Acute MyocardialInfarction (AMI) or chronically in patients with progressive heartfailure to stop the progression and worsening of the symptoms of CHF.

Hemodynamic Improvements:

Ventricular Pressure Reductions:

Treatment with TB4 showed a trend in improvement (i.e., a reduction) ofleft ventricular systolic pressure (LVSP) following heart muscledamage/injury. Hypertension and/or increased ventricular systolicpressures are/is an underlying cause of certain types of congestiveheart failure.

Treatment with TB4 significantly reduced left ventricular end diastolicpressure (LVEDP). Hypertension and/or elevated ventricular end diastolicpressures are/is a cause of certain type of congestive heart failure.

Ventricular Volume Reduction

Treatment with TB4 significantly attenuated left ventricular endsystolic volume, which is elevated in patients with CHF. Because endsystolic volume is a measurement of the adequacy of cardiac emptyingrelated to systolic function, it is associated with CHF patients withsystolic heart failure.

Treatment with TB4 significantly attenuated left ventricular enddiastolic volume. In the diseased heart, cardiac output falls ifventricular end diastolic volume rises to high levels as is the case inheart failure. Left ventricular end diastolic volume increases areassociated with CHF patients with diastolic heart failure.

Treatment with TB4 significantly improved rate of change (velocity) inleft ventricular pressure-rise with time (dP/dt), a surrogate measure ofventricular contractility. Because there is only a finite period of timeavailable for an ejection, a reduced ventricular pressure-rise rate ofvelocity (i.e., a longer time to contraction) results in less bloodejected per stroke. This is evidenced in CHF patients with systolicheart failure.

Treatment with TB4 showed a trend in affecting (improving) the rate ofchange (velocity) in left ventricular relaxation. The more negative thedP/dt the faster the heart is able to relax. CHF patients with diastolicheart failure have hearts that are less compliant, when relaxing andfiling with blood. This impedes blood filling into the ventricles andproduces backup into the lungs.

Organ Weights

Treatment with TB4 significantly attenuated pulmonary edema followingheart muscle damage/injury. Pulmonary edema is a serious outcome thatoccurs in patients with CHF as a result of left-sided heart failure.

Treatment of TB4 significantly attenuated thickening muscle of the leftventricle that negatively affect the pumping action of the heart.

In this study, organ weights (heart and lung) in the TB4-treated,vehicle-treated and untreated animal (sham) groups were assessed. Thesedata showed that TB4 attenuated both thickening of the heart muscle dueto stress/injury and pulmonary edema (fluid build up in the lung due toleft-sided heart failure), strongly indicating that TB4 may be effectivein treating patients with congestive heart failure (CHF). Since CHF(also called heart failure) is almost always a chronic, long-termcondition, chronic treatment by TB4 may retard or halt the progressionof a worsening condition.

EXAMPLE 2

The use of thymosin beta 4 was studied during the development ofdystrophic cardiomyopathy. We used the naturally occurring dystrophindeficient mdx mouse model and followed the cardiac functionlongitudinally with non-invasive echocardiography. Thymosin beta 4 mayhave beneficial effects on slowing the progression of the cardiomyopathythrough its properties of membrane stabilization and anti-fibrosis. Indystrophic cardiomyopathies, shear forces are poorly tolerated due tothe lack of the dystrophin and its connections to the extracellularmatrix. These forces lead to tearing of the muscle cell membranesleading to cell death and fibrosis. Thymosin beta 4 has been shown tohave membrane stabilizing properties, likely related to effects on actinpolymerization. We also followed functional parameters of skeletalmuscle function that may also benefit from administration of thymosinbeta 4. Preliminary data is presented in table 1. Thymosin beta 4 alsohas anti-fibrotic properties. Cardiac muscle injured due to shear forcesand calcium influx may benefit from thymosin beta 4's modulation ofmuscular remodeling. Less fibrosis maintains cardiac function for alonger period of time.

Test Parameters and Experimental Design:

Four groups of mice were treated with thymosin beta 4. Group 1 wasnormal mice (BL10) that were given placebo (untreated). Group 2 wasnormal mice that were treated with thymosin beta 4. Group 3 wasdystrophin deficient (mdx) mice treated with placebo and Group 4 was mdxmice treated with thymosin beta 4. Mice were treated with 150 microgramsof thymosin beta 4 in 300 microliters of buffer given intraperitoneallytwice a week and placebo mice were given 300 microliters of buffer only.The mice exercised on a treadmill at a speed of 12 meters/minute for 30minutes twice a week. Functional, behavioral and echocardiographic datawere obtained at baseline and after 2 months, 4 months and 6 months oftreatment.

Functional data were non-invasive measurements of skeletal musclefunction. The muscle strength of the forelimbs and hindlimbs wasassessed using a grip strength meter. The animals were allowed to holdonto the meter platform and then were slowly pulled until they losttheir grip. The amount of force needed to free the grip was recorded.Multiple measurements were taken for each mouse at each time point. Thesame protocol was followed for hindlimb muscle strength. Anotherfunctional assessment was the Rotarod test. Here, mice were placed on arotating bar and kept their balance and position as the bar rotated withincreasing speed (10 RPM for 1 minute and then increased 0.2 RPM overthe next three minutes). The time until the mouse falls was measured.This test was also repeated multiple times.

Behavioral data was collected using the VERSAMAX™ animal activitymonitoring system. Mice were placed in a monitored box and activity wasquantified by different sensors. Data includes horizontal activity,vertical activity and total distance traveled and many other activityparameters were measured. Multiple measurements were made over a 3 dayperiod.

Echocardiographic assessment was performed using the VISUALSONICS VEVO660™ high frequency system. Evaluation of cardiac chamber size,ventricular function and inflow/outflow Doppler velocities werecompleted under isoflorane anesthesia. The cardiologist performing andmeasuring the echocardiograms was blinded to study groups.

TABLE 1 Functional parameters measured in normal (BL10) and dystrophindeficient (mdx) mice at baseline and after two months of treatment withthymosin beta 4. Group BL10 BL10 BL10 BL10 Mdx Mdx Mdx Mdx untreateduntreated treated treated untreated untreated treated treated TimeBaseline 2 month Baseline 2 month Baseline 2 month Baseline 2 month Avgmax 5.57 6.10 5.57 5.73 4.46 4.07 4.46 3.78 forelimb strength (KGF/kg)Avg max 7.35 9.01 7.35 9.18 6.29 7.54 6.29 7.29 hindlimb strength(KGF/kg) Avg 1761 1302 1761 1287 1071 875 1071 961 horizontal activityAvg 28 15.6 28 13.1 12 9.0 12 8.2 vertical activity Avg total 375 252375 239 209 162 209 167 distance (cm) Avg 41 28.4 41 27 25 19.2 25 18.9movement time (s) Rotorod 214 170 214 191 211 172 211 147

1. A method of treatment for treating, preventing, inhibiting orreducing tissue deterioration, injury or damage due to congestive heartfailure disease, or for restoring tissue adversely affected by saiddisease, in a subject, comprising administering to a subject havingcongestive heart failure disease an effective amount of a compositioncomprising a peptide agent comprising amino acid sequence LKKTET (SEQ IDNO: 1) or a conservative variant thereof, LKKTNT (SEQ ID NO: 2) or aconservative variant thereof, Thymosin β4 (Tβ4), a Tβ4isoform, KLKKTET(SEQ ID NO: 3), LKKTETQ (SEQ ID NO: 4), oxidized Tβ4, an N-terminalvariant of Tβ4, a C-terminal variant of Tβ4, Tβ4^(ala,) Tβ9, Tβ10, Tβ11,Tβ12, Tβ13, Tβ14, Tβ15, gelsolin, vitamin D binding protein (DBP),profilin, cofilin, depactin, DNasel, vilin, fragmin, severin, cappingprotein, β-actinin or acumentin, in said tissue, so as to inhibit saidtissue deterioration, injury or damage due to said disease, or restoretissue adversely affected by said disease, wherein administration ofsaid peptide agent results in at least one of a reduction of leftventricular systolic pressure (LVSP) of a heart, reduction in leftventricular end diastolic pressure (LVEDP) of a heart, attenuation ofleft ventricular end systolic volume of a heart, attenuation of leftventricular end diastolic volume of a heart, an increase in rate ofchange (velocity) in left ventricular pressure-rise with time (dP/dt) ofa heart, an increase in rate of change (velocity) in left ventricularpressure decrease with time (pdP/dt) of a heart, attenuation ofpulmonary edema following heart muscle damage/injury, or attenuation ofmuscle thickness of a heart left ventricle.
 2. The method of claim 1,wherein said tissue deterioration, injury or damage comprises at leastone of an increase in heart size or mass, increase in size or mass ofheart tissue, thickening of heart muscle, or pulmonary edema.
 3. Themethod of claim 2 wherein said peptide agent is Tβ4.
 4. The method ofclaim 2 wherein said peptide agent is other than Tβ4.
 5. The method ofclaim 2 wherein said peptide agent is other than oxidized Tβ4.
 6. Themethod of claim 4 wherein said peptide agent comprises amino acidsequence KLKKTET (SEQ ID NO: 3), amino acid sequence LKKTETQ (SEQ IDNO:4), and N-terminal variant of Tβ4, a C-terminal variant of Tβ4, or anisoform of Tβ4.
 7. The method of claim 2 wherein said peptide agent isadministered to said subject at a dosage within a range of about 1-30micrograms.
 8. The method of claim 2 wherein said agent is administeredby direct administration to said tissue, or by intravenous,intraperitoneal, intramuscular, subcutaneous, inhalation, transdermal ororal administration, to said subject.
 9. The method of claim 2 whereinsaid composition is administered systemically.
 10. The method of claim 2wherein said composition is administered directly.
 11. The method ofclaim 2 wherein said composition is in a form of a solution, gel, cream,paste, lotion, spray, suspension, dispersion, salve, hydrogel, ointment,foam or oil.
 12. The method of claim 2 wherein said peptide agent is arecombinant or synthetic peptide.
 13. A method for screening for astimulating agent that stimulates production of a peptide agentcomprising amino acid sequence LKKTET (SEQ ID NO: 1) or a conservativevariant thereof, LKKTNT (SEQ ID NO: 2) or a conservative variantthereof, Thymosin β4 (Tβ4), a Tβ4isoform, KLKKTET (SEQ ID NO: 3),LKKTETQ (SEQ ID NO: 4), oxidized Tβ4, an N-terminal variant of Tβ4aC-terminal variant of Tβ4, Tβ4^(ala,) Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14,Tβ15, gelsolin, vitamin D binding protein (DBP), profilin, cofilin,depactin, DNasel, vilin, fragmin, severin, capping protein, β-actinin oracumentin, comprising contacting a tissue exhibiting congestive heartfailure disease, with a candidate compound; and measuring activity insaid tissue of said peptide agent, wherein an increase of activity ofsaid peptide agent in said tissue, compared to a level of activity ofsaid peptide agent in a corresponding tissue lacking said candidatecompound, indicates that said compound is capable of stimulatingproduction of said peptide agent.
 14. The method of claim 13 whereinsaid peptide agent is thymosin beta
 4. 15. The method of claim 1,wherein said peptide agent is administered acutely, chronically, or acombination thereof.